Iron occurs in oxidation states from −2 to +6 depending on both pH and the nature of the ligating groups surrounding the metal [Bergeron, R. J.; Brittenham, G. M. The Development of Iron Chelators for Clinical Use. CRC: Boca Raton, Fla., 1994]. It is these dependencies that nature has exploited so effectively in enlisting the metal as a central component in a myriad of redox processes [Bergeron, R. J.; McManis, J. S.; Wiegand, J.; Weimar, W. R. A Search for Clinically Effective Iron Chelators. In Iron Chelators: New Development Strategies, Badman, D. G.; Bergeron, R. J.; Brittenham, G. M., Eds. Saratoga: Ponte Vedra Beach, Fla., 2000; pp 253-292]. In fact, life without iron is virtually nonexistent [Crichton, R. R. Inorganic Biochemistry of Iron Metabolism. J. Wiley & Sons: Chichester, UK, 2001]. However, while the metal composes some 5% of the earth's crust, it is nevertheless difficult for living systems to access. In the biosphere, iron exists largely as Fe (III), in a variety of water insoluble forms, at pH 7. The concentration of free Fe(III) under these conditions is ≈1.4×10−9 M [Chipperfield, J. R.; Ratledge, C. Salicylate Is Not a Bacterial Siderophore: A Theoretical Study. BioMetals 2000, 13, 165-168], somewhat lower than that required to support most life forms. In the presence of phosphate ions in culture media and potential animal hosts, the free Fe(III) concentration in solution drops even further, by a factor of 10.
Both prokaryotes and eukaryotes have overcome the problem of iron accessibility by developing iron-binding ligands and associated transport systems [Bernier, G.; Girijavallabhan, V.; Murray, A.; Niyaz, N.; Ding, P.; Miller, M. J.; Malouin, F. Desketoneoenactin-Siderophore Conjugates for Candida: Evidence of Iron Transport-Dependent Species Selectivity. Antimicrob. Agents Chemother. 2005, 49, 241-248; Walz, A. J.; Mollmann, U.; Miller, M. J. Synthesis and Studies of Catechol-Containing Mycobactin S and T Analogs. Org. Biomol. Chem. 2007, 5, 1621-1628; Yuan, W. M.; Gentil, G. D.; Budde, A. D.; Leong, S. A. Characterization of the Ustilago maydis sid2 Gene, Encoding a Multidomain Peptide Synthetase in the Ferrichrome Biosynthetic Gene Cluster. J. Bacteriol. 2001, 183, 4040-4051; Byers, B. R.; Arceneaux, J. E. Microbial Iron Transport: Iron Acquisition by Pathogenic Microorganisms. Met. Ions Biol. Syst. 1998, 35, 37-66; Kalinowski, D. S.; Richardson, D. R. The Evolution of Iron Chelators for the Treatment of Iron Overload Disease and Cancer. Pharmacol Rev. 2005, 57, 547-583; Bergeron, R. J. Iron: A Controlling Nutrient in Proliferative Procsses. Trends Biochem. Sci. 1986, 11, 133-136; Theil, E. C.; Huynh, B. H. Ferritin Mineralization: Ferroxidation and Beyond. J. Inorg. Biochem. 1997, 67, 30 and Ponka, P.; Beaumont, C.; Richardson, D. R. Function and Regulation of Transferrin and Ferritin. Semin. Hematol. 1998, 35, 35-54]. Prokaryotes produce a group of iron chelators or siderophores (generally low-molecular weight, iron-specific ligands) that they secrete into the environment. These ligands often present with very large formation constants (e.g., 1048 M−1) [Bergeron, R. J.; McManis, J. S. Synthesis of Catecholamide and Hydroxamate Siderophores. In CRC Handbook of Microbial Iron Chelates, Winkelmann, G., Ed. CRC: Boca Raton, 1991; pp 271-307] and can effectively remove the metal from other donor arrays. The resulting metal complex, a ferrisiderophore, is then taken up by microorganisms [Ratledge, C.; Dover, L. G. Iron Metabolism in Pathogenic Bacteria. Annu. Rev. Microbiol. 2000, 54, 881-941; Nicholson, M. L.; Beall, B. Disruption of tonB in Bordetella bronchiseptica and Bordetella pertussis Prevents Utilization of Ferric Siderophores, Haemin, and Haemoglobin as Iron Sources. Microbiology 1999, 145, 2453-2461 and Occhino, D. A.; Wyckoff, E. E.; Hernderson, D. P.; Wrona, T. J.; Payne, S. M. Vibrio cholerae Iron Transport: Haem Transport Genes Are Linked to One of Two Sets of tonB, exbB, exbD Genes. Mol. Microbiol. 1998, 29, 1493-1507], most often beginning with binding to an outer membrane receptor [Stojiljkovic, I.; Srinivasan, N. Neisseria meningitidis tonB, exbB, and exbD Genes: Ton-Dependent Utilization of Protein-Bound Iron in Neisseriae. J. Bacteriol. 1997, 179, 805-812]. This is followed by shuttling the iron complex through the periplasm and finally, to the cytoplasm, where the iron is freed up. These ferrisiderophore transporters are energy-dependent, often exploiting the tonB system [Griffiths, E.; Williams, P. H. The Iron-Uptake Systems of Pathogenic Bacteria, Fungi, and Protozoa. 2 ed.; John Wiley & Sons: Chichester, UK, 1999; Ochsner, U. A.; Johnson, Z.; Vasil, M. L. Genetics and Regulation of Two Distinct Haem-Uptake Systems, phu and has, in Pseudomonas aeruginosa. Microbiology 2000, 146, 185-198 and Stoebner, J. A.; Payne, S. M. Iron-Regulated Hemolysin Production and Utilization of Heme and Hemoglobin by Vibrio cholerae. Infect. Immun. 1988, 56, 2891-2895]. In most instances, the desferrisiderophore is released to further gather iron.
There have now been over 500 different siderophores identified [Drechsel, H.; Winkelmann, G. Iron Chelation and Siderophores. In Transition Metals in Microbial Metabolism, Winkelmann, G., Carrano, C. J., Eds. Harwood Acad.: Amsterdam, the Netherlands, 1997; pp 1-9; Neilands, J. B. Siderophores: Structure and Function of Microbial Iron Transport Compounds. J Biol. Chem. 1995, 270, 26723-26726; Telford, J. R.; Raymond, K. N. Siderophores. In Comprehensive Supramolecular Chemistry, Atwood, J. L., Davies, J. E. D., MacNicol, D. D., Vogtle, F., Lehn, J-M., Eds. Elsevier Sci.: Oxford, UK, 1996; Vol. 1, pp 245-266; Winkelmann, G. CRC Handbook of Microbial Iron Chelates. CRC: Boca Raton, Fla., 1991 and Winkelmann, G.; Drechsel, H. Microbial Siderophores. Biotechnology. 2 ed.; Verlag Chem.: Weinheim, Germany, 1997; Vol. 7]. While there are certainly exceptions, the two main classes of natural product iron chelators are hydroxamates [Bergeron, R. J.; Phanstiel, O., IV. The Total Synthesis of Nannochelin: A Novel Cinnamoyl Hydroxamate-Containing Siderophore. J. Org. Chem. 1992, 57, 7140-7143; Bergeron, R. J.; McManis, J. S. Synthesis and Biological Activity of Hydroxamate-Based Iron Chelators. In The Development of Iron Chelators for Clinical Use, Bergeron, R. J.; Brittenham, G. M., Eds. CRC: Boca Raton, 1994; pp 237-273; Bergeron, R. J.; McGovern, K. A.; Channing, M. A.; Burton, P. S. Synthesis of N4-Acylated N1,N8-Bis(Acyl)Spermidines: An Approach to the Synthesis of Siderophores. J. Org. Chem. 1980, 45, 1589-1592; Bergeron, R. J.; Kline, S. J. Short Synthesis of Parabactin. J. Am. Chem. Soc. 1982, 104, 4489-4492; Bergeron, R. J.; McManis, J. S.; Dionis, J. B.; Garlich, J. R. An Efficient Total Synthesis of Agrobactin and Its Gallium(III) Chelate. J Org. Chem. 1985, 50, 2780-2782 and Bergeron, R. J.; Garlich, J. R.; McManis, J. S. Total Synthesis of Vibriobactin. Tetrahedron 1985, 41, 507-510], such as desferrioxamine (1) and catecholamides [Bergeron, R. J.; Pegram, J. J. An Efficient Total Synthesis of Desferrioxamine. J. Org. Chem. 1988, 53, 3131-3134; Bergeron, R. J.; McManis, J. S. The Total Synthesis of Desferrioxamines E and G. Tetrahedron 1990, 46, 5881-5888; Bergeron, R. J. Synthesis and Solution Structures of Microbial Siderophores. Chem. Rev. 1984, 84, 587-602; Bergeron, R. J.; McManis, J. S. Total Synthesis of Bisucaberin. Tetrahedron 1989, 45, 4939-4944 and Bergeron, R. J.; McManis, J. S.; Perumal, P. T.; Algee, S. E. The Total Synthesis of Alcaligin. J. Org. Chem. 1991, 56, 5560-5563], including vulnibactin (2) and vibriobactin (3) (FIG. 1). Some microorganisms can, in fact, utilize more than one type and/or class of siderophores [Kim, C.-M.; Park, Y.-J.; Shin, S.-H. A Widespread Desferrioxamine-Mediated Iron-Uptake System in Vibrio vulnificus. J. Infect. Dis. 2007, 196, 1537-1545].
Iron acquisition becomes somewhat more problematic for microorganisms in an in vivo situation (e.g., in humans). Pathogens have additional iron acquisition hurdles to overcome beyond low metal solubility. Animals, for example, have an iron-withholding system: proteinaceous iron chelators that make iron acquisition difficult for microorganisms. There is little of the free metal available in animals. It is generally bound to heme (iron-containing enzymes) by transferrin, (an iron shuttle protein) or stored in ferritin. In each instance, iron is not easily accessible to microorganisms.
The opportunistic microorganism Vibrio vulnificans nicely illustrates how pathogens can overcome host iron-withholding [Stoebner, J. A.; Butterton, J. R.; Calderwood, S. B.; Payne, S. M. Identification of the Vibriobactin Receptor of Vibrio cholerae. J. Bacteriol. 1992, 174, 3270-3274]. The siderophore produced by Vibrio vulnificans, vulnibactin (2) (FIG. 1), cannot remove iron from transferrin, the ever-present iron shuttle protein in plasma, in spite of the fact 2 binds iron more tightly than transferrin. The chelator cannot access transferrin iron, as it is bound within the protein.
To solve this problem, the microorganism secretes a protease, which cleaves transferrin, thus releasing iron. The metal is then sequestered by 2, and the ferrisiderophore is taken up via an intermembrane receptor, viuA [Butterton, J. R.; Stoebner, J. A.; Payne, S. M.; Calderwood, S. B. Cloning, Sequencing, and Transcriptional Regulation of vuuA, the Gene Encoding the Ferric Vibriobactin Receptor of Vibrio cholerae. J. Bacteriol. 1992, 174, 3729-3738; Simpson, L. M.; Oliver, J. D. Siderophore Production by Vibrio vulnificus. Infect. Immun. 1983, 41, 644-649 and Okujo, N.; Akiyama, T.; Miyoshi, S.; Shinoda, S.; Yamamoto, S. Involvement of Vulnibactin and Exocellular Protease in Utilization of Transferrin- and Lactoferrin-Bound Iron by Vibrio vulnificus. Microbiol. Immunol. 1996, 40, 595-598].
In fact, Vibrio vulnificans mutants without the vulnibactin transporter have reduced pathogenicity in mice [Webster, A. C. D.; Litwin, C. M. Cloning and Characterization of vuuA, a Gene Encoding the Vibrio vulnificus Ferric Vulnibactin Receptor. Infect. Immun. 2000, 68, 526-534]. This uptake apparatus has been shown to have significant homology with the Vibrio cholerae receptor. However, while it seems clear from studies with genetically altered microorganisms that shutting down the siderophore iron-uptake system can slow growth and reduce pathogenicity, microorganisms can still access iron via other mechanisms [Henderson, D. P.; Payne, S. M. Vibrio cholerae Iron Transport Systems: Roles of Heme and Siderophore Iron Transport in Virulence and Identification of a Gene Associated with Multiple Iron Transport Systems. Infect. Immun. 1994, 62, 5120-5125; Litwin, C. M.; Rayback, T. W.; Skinner, J. Role of Catechol Siderophore in Vibrio vulnificus Virulence. Infect. Immun. 1996, 64, 2834-2838 and Stelma, G. N.; Reyes, A. L.; Peeler, J. T.; Johnson, C. H.; Spaulding, P. L. Virulence Characteristics of Clinical and Environmental Isolates of Vibrio vulnificus. Appl. Environ. Microbiol. 1992, 58, 2776-2782]. For example, Vibrio cholerae can utilize transferrin and heme as iron sources. The issue then becomes how useful a target the siderophore transport apparatus is in antimicrobial design strategies.
Miller has, in a series of classic studies, employed siderophores and the corresponding transporters as vectors for the delivery of antibiotics [Miller, M. J.; Malouin, F. Siderophore-Mediated Drug Delivery: The Design, Synthesis, and Study of Siderophore-Antibiotic and Antifungal Conjugates. In The Development of Iron Chelators for Clinical Use, Bergeron, R. J.; Brittenham, G. M., Eds. CRC: Boca Raton, 1994; pp 275-306]. Alternatively, Esteve-Gassent was able to demonstrate that a vaccine developed to treat eels infected with Vibrio vulnificus serovar E. contained antigens to the putative receptor for vulnibactin. Esteve-Gassent point out that the antibody could be blocking siderophore uptake, could trigger classical complement activation, or “mark bacteria for opsonophagocytosis.” [Esteve-Gassent, M. D.; Amaro, C. Immunogenic Antigens of the Eel Pathogen Vibrio vulnificus Serovar E. Fish Shellfish Immunol. 2004, 17, 277-291].